It has been reported that collagen II (CII) derived peptide CII263-272 induced T-cell activation via its amino acids responsible for T-cell receptor (TCR) recognition. The impact of substitution of the TCR contacting amino acids of CII263-272 on T-cell activation was evaluated in this study using a panel of altered CII263-272 peptides. Computer modeling revealed that the side chains of 263F and 266E in CII263-272 were coupled with amino acids on 1 and 1 chains of HLADR1 or-DR4, mainly via hydrogen bonds, whereas the side chains of 267Q and 270K protrude out of the cleft and might be recognized by TCR. Intracellular delivery of the altered peptides, and their binding to HLA-DR1 and DR4 molecules on cell surface, were demonstrated by confocal microscopy and flow cytometry. The results also revealed that the substitution of 267Q, 268G, 269P, and 270K individually or consecutively by alanine (A) or glycine (G) led to weak or non-T-cell responses. Furthermore, the altered peptides with 270K substitution (270A) or with consecutive substitution of 268G, 269P, and 270K (sub268-270) dramatically inhibited T-cell activation. It is suggested that the altered peptides derived from CII263-272 with substitution of amino acids responsible for TCR contact might be of inhibitory effect on T-cell responses.